Sixteenth century wall paintings were analyzed from a church in an advanced state of decay in the Apennines of central Italy, now a remote area but once located along the salt routes from the Po Valley to the Ligurian Sea. Infrared spectroscopy (FTIR-ATR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) with a microprobe were used to identify the painting materials, as input for possible future restoration. Together with the pigments traditionally used for wall painting, such as ochre, ultramarine blue, bianco di Sangiovanni, cinnabar/vermilion, azurite, some colors were also found to have only been used since the 18th century. This thus suggests that a series of decorative cycles occurred after the church was built, confirmed by the multilayer stratigraphy of the fragments. Some of these colors were also unusual, such as clinochlore, Brunswick green, and ultramarine yellow. The most notable result of the analytical campaign however, was the ubiquitous determination of aragonite, the mineralogical form of calcium carbonate, mainly of biogenic origin. Sources report its use in Roman times as an aggregate in mortars, and in the literature it has only been shown in Roman wall paintings. Its use in 16th century wall paintings is thus surprising.